1. Field of the Invention
The present invention relates generally to optically transduced assays performed on waveguides and more specifically to assays performed on multimode waveguides.
2. Description of the Background Art
Throughout the present specification, all referenced papers and patents are incorporated herein by reference for all purposes.
In pursuit of the goal of multi-analyte sensing, a variety of methods for immobilizing recognition molecules in arrays and devices for interrogating these arrays have been reported. The array biosensors designed for this goal can be divided into two groups with regard to the method of immobilization and type of molecules immobilized. In the approach pioneered by Fodor and colleagues (Fodor et al., Science, 251 767 (1991)), photolithographic activation is used to build up polymers of nucleotides or amino acids in arrays of 1024 elements. These binding molecules are relatively short and only semi-selective: thus, pattern recognition is required for detection. Such systems are becoming widely used for genetic and drug screening where the user wants to screen for a large number of functions or "matches" simultaneously. In the second approach, fully formed molecules such as antibodies or longer nucleic acid strands are attached to the surface (Rowe et al., Anal. Chem. 71:(2) 433-439 Jan. 15, 1999; Conrad et al., U.S. Pat. No. 5,736,257; Conrad et al., SPIE, 2978, p. 12 (1997); Wadkins et al., Biosensors & Bioelectronics, 13, 407 (1998); Martin et al., Micro Total Analysis Systems '98 (Kluwer Academic Publishers, Netherlands, 1998) p. 27). Due to the high specificity of binding, a detection event at a single spot is sufficient for identification. These systems are of more interest to users who want to screen for moderate numbers (i.e., 3-500) of previously identified analytes.
The types of optical devices fabricated for interrogation of these two types of arrays also differ. Instruments for analysis of large numbers of very small elements must have relatively high resolution and are usually based on confocal microscopy. They may often include fairly sophisticated image analysis and pattern recognition software to interpret the semi-selective element responses. Size and weight are not major issues as these devices are intended for use in a laboratory. Instruments for detecting signals from arrays of the second type tend to be geared for portability and low cost (Wadkins et al. , supra; Ligler et al., 1998; Herron et al., 1996; Herron et al., 1997; Katerkamp 1997; Duveneck et al., 1995; Duibendorfer and Kunz 1998). The emphasis in this case is on gearing the element size to the device rather than the opposite. The array biosensor described here falls in this latter category and is targeted for use at the bedside for clinical testing, or outdoors for environmental monitoring.
Optical waveguides are advantageous for portable and low cost instruments. In such devices, excitation light traveling in an optical waveguide is confined by total internal reflection (TIR) at the interface defined by the waveguide surface. TIR occurs only under a limited set of conditions and is dependent on a number of factors, including the wavelength, incidence angle, and the relative refractive indices of the waveguide and the surrounding medium (Axelrod et al., Ann. Rev Biophys. Bioeng, 13, 247 (1984)). The surrounding medium, referred to as the "cladding", must be of a lower refractive index than the waveguide in order to achieve TIR.
Functionally, designing a system around TIR becomes complex when the goal is not to simply confine light within the waveguide but to use the non-radiative evanescent field generated at the interface to probe material on the waveguide surface. For sensors of this type, it is beneficial to optimize the strength of the evanescent field by maximizing its penetration depth, d.sub.p. Given a waveguide and cladding pair of fixed refractive indices, the penetration depth of the evanescent field increases as the optical incidence angle decreases towards the critical angle. The relation is defined by: d.sub.p.varies.((n.sub.2 /n.sub.1).sup.2 sin.sup.2 .phi.-1).sup.-1/2, where .phi. is the angle measured from the surface normal and n.sub.1 and n.sub.2 are the refractive indexes of the surrounding medium and waveguide, respectively, and n.sub.2 &lt;n.sub.1 (Love et al., SPIE, 990, p. 175 (1988)). However, TIR will not occur beyond a critical angle as derived from Snell's law, where sin .phi..sub.critical =n.sub.1 /n.sub.2. The critical angle for an air-glass interface is approximately 42.degree. and is 67.degree. for a water-glass interface.
Thus, there is an inherent conflict between maintaining light by TIR and maximizing the evanescent field when a cladding is used on the waveguide. As suggested previously, this conflict becomes problematic when the waveguide will be used not only to confine and transmit light but, also to operate as an evanescent field excitation source or sensor in a low index environment, such as air (n=1) or water (n=1.33). Since appropriate cladding materials with refractive indices in the range between air and water do not readily exist, it is not possible both to maximize the evanescent field in air or water and to confine the light with a cladding by TIR.
This conflict could potentially be resolved by developing the sensor around a tapered waveguide or a mono-mode waveguide designed for an optimum balance between optical confinement and a evanescent field penetration depth (Anderson et al., Biosensors & Bioelectronics, 8, p. 249 (1993); Anderson et al., U.S. Pat. No. 5,430,813 (1995); Duveneck et al., Sensors & Actuators B, 38-39, 88 (1997)). However, it is preferable for a number of reasons to base the sensor on a multi-mode waveguide. First, multi-mode waveguides can be very simple and inexpensive. For example, the planar waveguides used in the present invention can be commercial quality microscope slides. On the other hand, mono-mode waveguides are typically formed by a precision film deposition method such as vacuum deposition. Such processing, especially given required tolerances on the order of tens of nanometers, yields a relatively expensive waveguide.
A second relative advantage of a multi-mode waveguide is that coupling light into the guide is trivial in comparison with a mono-mode waveguide. All that is required to couple light into the waveguide is to direct the light beam onto the end face at an angle within the waveguide's numerical aperture. Under these conditions, once the light enters the waveguide, it will be confined and guided by TIR. Further, given the relatively large face of a multi-mode waveguide, the positioning of the beam on the end face can vary significantly and still be efficiently coupled into the waveguide. A highly tolerant alignment such as this is ideally suited for a system to be used in the field.
In contrast, with a mono-mode waveguide, which is at most a few microns thick, it is not possible to use simple end-coupling to get light into the guide. Coupling in this manner is further complicated by the inherent multi-mode output of diode lasers since a multi-mode beam cannot be focused as tightly a mono-mode laser beam. Instead, coupling into a mono-mode waveguide is generally achieved using gratings or prisms. However, for a sensor system that is intended to be robust and portable, the very tight angular alignment tolerances inherent in using a grating or prism argue against the use of mono-mode waveguides.
The two-dimensional surface of the waveguide lends itself to spatial patterning of multianalyte array elements and image analysis using only a single wavelength and excitation source and fluorophore. This approach is inherently more flexible and less complicated than trying to resolve multiple wavelengths of different fluorophores within a single element, both in terms of the number of analytes that can be measured simultaneously and by the requirement for complex optics. To this end, a method for forming the spatially patterned sensing elements and the means of measuring signals from a large number of elements has been developed.
Another desirable feature for analytical devices, multi-sample processing, requires a large number of fluid connections and there are several considerations for connector design (Mourlas et al., Micro Total Analysis Systems '98 (Kluwer Academic Publishers, Netherlands, 1998) p. 27). First, manipulation of fluids and introduction of samples and reagents to the waveguide should be accomplished with minimal increase in the size of the sensor. In addition, fluid-tight attachments should be accomplished rapidly and simply so that little effort is required by the user to replace sensor elements and analyze additional samples. To this end, a means of attaching mounting brackets to the waveguide-flow cell combination has been developed.
Although the waveguide choice influences many of the patterning and fluidics methods, system development is an iterative process and the choice of fluidics feeds back into the design of the waveguide. Specifically, the problem of attaching a fluidics cell to the waveguide without perturbing the confinement of the excitation light, but still allowing for optimization of the evanescent field in either air or water, must be resolved.